It is proposed here that the delayed cytotoxicity of thioguanine involves the postreplicative DNA mismatch repair system. After incorporation into DNA, the thioguanine is chemically methylated by S-adenosylmethionine to form S6-methylthioguanine. During DNA replication, the S6-methylthioguanine directs incorporation of either thymine or cytosine into the growing DNA strand, and the resultant S6-methylthioguanine-thymine pairs are recognized by the postreplicative mismatch repair system. Azathioprine, an immunosuppressant used in organ transplantation, is partly converted to thioguanine. Because the carcinogenicity of N-nitrosamines depends on formation of O6-alkylguanine in DNA, the formation of the analog S6-methylthioguanine during azathioprine treatment may partly explain the high incidence of cancer after transplantation.
In vitro, following the removal of thymine from a G⅐T mismatch, thymine DNA glycosylase binds tightly to the apurinic site it has formed. It can also bind to an apurinic site opposite S 6 -methylthioguanine ( SMe G) or opposite any of the remaining natural DNA bases. It will therefore bind to apurinic sites formed by spontaneous depurination, chemical attack, or other glycosylases. In the absence of magnesium, the rate of dissociation of the glycosylase from such complexes is so slow (k off 1.8 ؊ 3.6 ؋ 10 ؊5 s ؊1 ; i.e. half-life between 5 and 10 h) that each molecule of glycosylase removes essentially only one molecule of thymine. In the presence of magnesium, the dissociation rates of the complexes with C⅐AP and SMe G⅐AP are increased more than 20-fold, allowing each thymine DNA glycosylase to remove more than one uracil or thymine from C⅐U and SMe G⅐T mismatches in DNA. In contrast, magnesium does not increase the dissociation of thymine DNA glycosylase from G⅐AP sites sufficiently to allow it to remove more than one thymine from G⅐T mismatches. The bound thymine DNA glycosylase prevents human apurinic endonuclease 1 (HAP1) cutting the apurinic site, so unless the glycosylase was displaced, the repair of apurinic sites would be very slow. However, HAP1 significantly increases the rate of dissociation of thymine DNA glycosylase from apurinic sites, presumably through direct interaction with the bound glycosylase. This effect is concentration-dependent and at the probable normal concentration of HAP1 in cells the dissociation would be fast. This interaction couples the first step in base excision repair, the glycosylase, to the second step, the apurinic endonuclease. The other proteins involved in base excision repair, polymerase , XRCC1, and DNA ligase III, do not affect the dissociation of thymine DNA glycosylase from the apurinic site.In mammalian cells, 2-7% of the total cytosine is methylated. Spontaneous deamination of 5-methylcytosine, which is somewhat faster than cytosine (1), generates G⅐T mispairs in DNA. The repair of these G⅐T mismatches is initiated by thymine DNA glycosylase which excises the mismatched thymine (2, 3). 5-Methylcytosine occurs almost exclusively in the sequence Me CpG, and in keeping with its proposed role in the repair of G⅐T mispairs resulting from the deamination of 5-methylcytosine, thymine DNA glycosylase shows a strong preference for removal of thymine from CpG⅐T sequences (4 -7). The human enzyme has been cloned and overexpressed (8) and has been shown to belong to a family of uracil DNA glycosylases that remove uracil from G⅐U base pairs but that are distinct from the general uracil DNA glycosylase enzyme (9). Thymine DNA glycosylase removes uracil from G⅐U base pairs more rapidly than it removes thymine from G⅐T base pairs (10) and can also remove uracil from C⅐U, T⅐U, and A⅐U base pairs (7) and may therefore provide a backup function to the general uracil DNA glycosylase. The glycosylase also removes thymine from base pairs with S 6 -methylthioguanine ( SMe G) 1 that ar...
The time course of removal of thymine by thymine DNA glycosylase has been measured in vitro. Each molecule of thymine DNA glycosylase removes only one molecule of thymine from DNA containing a G⅐T mismatch because it binds tightly to the apurinic DNA site left after removal of thymine. The 5-flanking base pair to G⅐T mismatches influences the rate of removal of thymine: k cat values with C⅐G, T⅐A, G⅐C, and A⅐T as the 5-base pair were 0.91, 0.023, 0.0046, and 0.0013 min ؊1 , respectively. Thymine DNA glycosylase can also remove thymine from mismatches with S 6 -methylthioguanine, but, unlike G⅐T mismatches, a 5-C⅐G does not have a striking effect on the rate: k cat values for removal of thymine from SMe G⅐T with C⅐G, T⅐A, G⅐C, and A⅐T as the 5-base pair were 0.026, 0.018, 0.0017, and 0.0010 min ؊1 , respectively. Thymine removal is fastest when it is from a G⅐T mismatch with a 5-flanking C⅐G pair, suggesting that the rapid reaction of this substrate involves contacts between the enzyme and oxygen 6 or the N-1 hydrogen of the mismatched guanine as well as the 5-flanking C⅐G pair. Disrupting either of these sets of contacts (i.e. replacing the 5-flanking C⅐G base pair with a T⅐A or replacing the G⅐T mismatch with SMe G⅐T) has essentially the same effect on rate as disrupting both sets (i.e. replacing CpG⅐T with Tp SMe G⅐T), and so these contacts are probably cooperative.The glycosylase removes uracil from G⅐U, C⅐U, and T⅐U base pairs faster than it removes thymine from G⅐T. It can even remove uracil from A⅐U base pairs, although at a very much lower rate. Thus, thymine DNA glycosylase may play a backup role to the more efficient general uracil DNA glycosylase.G⅐T mismatches are produced in DNA by replication errors and by the deamination of 5-methylcytosine. In human cells, errors from these two sources are probably repaired in different ways. The G⅐T mismatches from replication errors are thought to be repaired by the postreplicative mismatch repair pathway (1, 2), but the G⅐T mismatches from the deamination of 5-methylcytosine are repaired by base excision repair (reviewed in Refs. 3 and 4) initiated by excision of the thymine by thymine DNA glycosylase (5, 6). The strong preference of thymine DNA glycosylase for G⅐T mismatches in the sequence CpG⅐T (7-10) is consistent with the view that the role of thymine DNA glycosylase is to remove thymine produced through deamination of 5-methylcytosine, because cytosine is methylated almost exclusively in CpG sequences. Although thymine DNA glycosylase can remove uracil as well as thymine (11), cloning of the human enzyme (12) showed that it has no sequence homology to the general uracil DNA glycosylase (EC 3.2.2.3). However, it is homologous to a class of uracil glycosylases specific for G⅐U mismatches (13). Because of their specificity for uracil in G⅐U mismatches, and to distinguish them from the general uracil DNA glycosylases, these mismatch-specific uracil glycosylases have been called mismatch-specific uracil DNA glycosylases (MUGs) 1 (14). Like thymine DNA glycosyl...
It has been suggested that the cytotoxicity of 6-thioguanine depends upon (1) incorporation of 6-thioguanine into DNA, (2) methylation by S-adenosylmethionine (SAM) of the thio group to give S6-methylthioguanine, (3) miscoding during DNA replication to give [SMeG] x T base pairs, and (4) recognition of these base pairs by proteins of the postreplicative mismatch repair system. Here we have investigated systematically the ability of proteins present in human cell extracts to bind to DNA containing S6-methylthioguanine. We found that [SMeG] x T base mismatches were bound by the mismatch binding complex, hMutS alpha, and that the level of binding was dependent upon the base 5' to the S6-methylthioguanine in the order G > C = A > T. Extracts from cells that lack either hMSH2 (LoVo cells) or GTBP (HCT-15 cells), two components of the hMutS alpha complex, were unable to bind the [SMeG] x T base pair. We also found that hMutS alpha was able to bind to [SMeG] x C base pairs when the S6-methylthioguanine was in the sequence 5'-Cp[SMeG]. This suggests that miscoding by S6-methylthioguanine residues in DNA during DNA synthesis may not be an absolutely required step in the mechanism of cytotoxicity. Also, since CpG sequences are so important in gene regulation, this result may be of considerable significance.
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